Many laboratories in the fields of biochemistry, chemistry, medicine, diagnostics and the like are interested in the measurement of generation of enzymes in biological media. To this end, signal substrates are generally used that release a chromophore or fluorophore and, thus, after conversion will yield a chromogenic or fluorogenic signal that can be followed in time and measured.
A convenient method to follow the formation of enzyme in time in a biological medium is to add the substrate directly into the biological medium, e.g. the addition of a fluorogenic substrate to clotting plasma in which thrombin is formed. This has the advantage that the enzyme generation is measured in its physiological medium.
Drawbacks of this method are that the activity of the enzyme towards any physiological substrate that may also be present in this medium will compete with the signal substrate that is added and that the substrate may be completely consumed before the enzyme generation has finished. To minimize these effects, substrates are usually selected that do not bind to the enzyme too tightly (i.e. having a low KM) and that are not converted too fast (i.e. having a low kcat).
Another problem is that the measured signal is usually dependent on the turbidity and the color of the medium in which the reaction takes place. The same amount of enzyme in media from different sources or donors may thus yield a different amount of signal. Also the chromophore or fluorophore concentration may not be directly proportional to the amount of signal. This makes it more difficult to calculate the amount of enzyme present during the time course of the reaction. In order to accurately quantify the concentration of enzyme in time, all these effects have to be taken into account.
WO 03/093831 discloses a method for determining in real time the course of proteolytic activity, in particular thrombin activity, in a sample of blood or plasma as it appears in and disappears from the sample which comprises adding a thrombin substrate to the sample that, per unit time, produces a detectable signal in a quantity that bears relation to the amount of thrombin present. Simultaneously, in a control sample of the same blood or plasma in which thrombin generation is not triggered, the activity of a standard preparation with invariable thrombin activity is measured. The exact molar amount of thrombin present at any moment is obtained by comparison of the activity measured in clotting blood with a calibrator curve obtained from the simultaneously measured calibrator. It is also disclosed that if the color of the medium does influence the signal, then this calibrator curve can best be determined in each color of medium that is used.
The method of measuring the same amount of calibrated enzyme in another medium of different color or turbidity will produce a very similar calibration curve except for so-called medium-dependent effects.
This invention provides an alternative method in which this calibration curve is measured only once in one medium and the measurements in similar media of different color or turbidity are no longer performed by measuring the whole curve but can be replaced by a “single-point” measurement.